June 13, 2017

Investigating Legionella: PCR Screening and Whole-Genome Sequencing

Kimberlee Musser, PhDChief, Bacterial DiseasesWadsworth Center

2

1976 Philadelphia Legionnaires' disease outbreak

The total number of cases reached 221, 34 had died.

Bellevue-Stratford HotelJuly 21-24, 1976

June 13, 2017 3

1992 International Symposium on Legionella , ASM Press, 1993

4

Reported Cases of Legionellosis by yearNew York

0

200

400

600

800

100020

0420

0520

0620

0720

0820

0920

1020

1120

1220

1320

1420

15

NYS LegionellosisNYC LegionellosisTotal

Year

# Ca

ses

5

Legionella Culture Archive

• 1643 L. pneumophila 1• 306 L. pneumophila 6• 176 L. pneumophila 5• 105 L. pneumophila 4• 105 L. anisa• 84 L. pneumophila 3• 50 L. micdadei• 42 L. pneumophila 12• 27 L. pneumophila 4/5• 24 L. feelei• 20 L. bozemanii• 133 Legionella species• 77 L. pneumophila no serotype

2945 archived (1982-2017)

*55 % Lp180 % Lp

June 13, 2017 6

PFGE Analysis

• 1990 implemented (27 yrs exp.)• 1990-2003 Lab notebooks, reports• 2003 Bionumerics Database 1477

– 1477 entries– 415 patterns

7

Investigating Outbreaks (1982-2017)

• Total of 668 investigation testing requests– Average 19 requests per year– 128 total facilities/locations– Spanning 1 time testing to multiyear sampling – (2-34 years)

8Historic Outbreaks with Sources Identified

Category Number

Hospital

Potable water 20

Potable water /Cooling tower 1

Cooling tower 2

Nursing home/assisted living

Potable water 10

Cooling tower 3

Private home Potable water 2

Hotel Potable water 1

Correctional Facility Potable water 2

Residential Complex Potable water 2

Cooling Tower 1

School Potable water 1

Cooling tower = 7, Potable water = 39

*36% success rate for determining source 46/128

9

Real-time PCR testing • Started testing in 1998 (19 yrs exp.)• Validations for clinical testing, NYS approvals• Side by side testing comparisons

Screen samples, confirm identification of isolates and detect in clinical specimens

– L. pneumophila serogroup 1– L. pneumophila (1-15)– Legionella spp.– Inhibition control

– L. anisa– L. micdadei

10

PCR +/Cult - PCR -/Cult +

11

Legionella Outbreak Communication

Lab

HAI Epi

BCDC Epi

Environmental Health

Legionella Case ReportsInterviewsMedical record reviewsInteraction with facility, physiciansCommunity or HAIRequest cultures, specimens

Assessment of facilityInteraction with facilityReview of maintenance recordsSampling recommendations

Discussing testing volumes, typesPreparing – ordering media, labellingTesting timelinesShipping, Testing info

📱📱📞📞📧📧

📱📱📞📞📧📧

📱📱📞📞📧📧

Regional Epi

12

Can whole-genome sequencing (WGS) provide

additional resolution to outbreak investigations?

13

Ongoing pilot project with the CDC Legionella Lab AMD funding-Spring 2015

• Build an analytic bioinformatics pipeline for WGS SNP-based detection

• Retrospectively sequence characterized Legionella pneumophila outbreaks

• Determined the ability of WGS at correctly separating unrelated outbreak clusters and have a baseline of cluster definition for future usage

14

2006-2007

2011-2012

Maximum parsimony tree, ref seq based on 24,424 Core SNP matrix

Results

2004-2006

2009

418 SNPs

15

NYS Facility Investigation• 4 patients diagnosed in Aug/Sept 2015• Water restrictions and remediation occurred• November 2015 facility letter final culture• January 2015 5th patient diagnosed

– No evidence that the patient spent any time in areas of facility where Legionella pneumophila serogroup 4 (Lp4) has been recovered from potable water and no distinct water exposure could be identified.

• Patient’s Lp4 isolate was indistinguishable by PFGE from other patients at facility and two environmental cultures from the potable water system

WGS initiated

16

PFGE and serotyping were performed on 4 patient isolates and 3 environmental samples

PFGE results indicated the isolates were all the same strain

All isolates were determined to be a serogroup 4*

5th patient

17WGS SNP Analysis

• The 5th patient was actually a closer match to Patient B, Patient A, and environmental sources than Patient C was.

• All patient and environmental samples in this investigation were 1-13 SNPs different when compared to the 5th patient and >23,000 SNPs different from any other of the >320 Legionella we have sequenced to date.

5th patient

5th patient

18

The Challenge of interpretation of SNP differences

• 1 SNP difference is meaningful• <10 SNPs, <5 SNPs indicate the same

source?• Heterogeneity in sources• Reference genome- when is this critical

for interpretation of an outbreak?

June 13, 2017 19Legionella Persistence in Facilities

20 SNPs different

20Legionella Whole-genome Sequencing

454 Legionella to date

21

WGS activities• Outbreak Investigations• Facility Persistence• CT Heterogeneity• Serogroup switching• Primary specimens• CDC AMD wgMLST

June 13, 2017 22

Liz NazarianDeb BakerDianna BoppNellie DumasAmy Saylors

Lisa MingleKara MitchellTeresa PassarettiDanielle WroblewskiWilliam Lainhart

Anna KidneyYan ZhuTanya HalseLisa ThompsonAcknowledgements

BACTERIOLOGY LABORATORY

BIODEFENSE LABORATORY

Bioinformatics Core- Pascal Lapierre

APPLIED GENOMIC TECHNOLOGIES CORE

CDC AMD Funding

Tissue and Media Core, Security, Director’s Office, DLO, CLIMs groupJill Taylor, Vicky Derbyshire, Ron Limberger, Colleen Walsh

NYC PHL- Jen Rakeman, Scott Hughes, Tony Tran, John Kornblum, Laura Kornstein, Ying Lin

NYC Epidemiology- Don Weiss, Robert Fitzhenry, Isaac Benowitz

NYSDOHNYS CEH/NYC CEH CDC Pneumonia Response and Surveillance Laboratory (NCIRD/DBD/RDB)- Brian Raphael, Natalia A. Kozak-Muiznieks , Shatavia S. Morrison , Claressa E. Lucas, Jeffrey W. Mercante , Jonas M. Winchell

Christina Egan, Michael Perry

Matt Shudt, Patrick Van Roey

24 Month Full-Time Intensive Program

Classes – 50% GPA

Laboratory Rotations – 20% GPA

Capstone Research – 30% GPA

Tuition and Scholarships No tuition/fees

Stipends awarded based on academic merit and funding availability

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